Summary Apicomplexan parasites are responsible for severe human diseases. Drug resistance and/or poor specificity are constantly undermining therapeutic regimens to treat these diseases. In order to identify new drug targets, the P.I.'s lab focuses on deepening the understanding of cell biological processes wherein the parasite differs from its host using Toxoplasma gondii as model apicomplexan. In this proposal they will address such distinct structure: the basal complex (BC), which sits at the posterior end of the unique cortical membrane skeleton of these parasites. Historically, interest in the BC stems from its function as the contractile ring driving cell division. Basal complex contraction is powered by an as yet undefined mechanism independent of actin- myosin setting it apart from the host. More recently, the BC has also been associated with other processes such as assembly of the tubulovesicular intravacuolar network (IVN), which operates as an exchanger between parasite and host cell and is additionally essential to establish a chronic infection. Contractile rings in other systems are composed of 125+ proteins, yet only 22 BC proteins are known. To decipher the molecular mechanisms behind the BC's diverse functions, it is proposed to assemble its complete parts list through an in vivo proximity-based biotinylation approach (BioID). Since BioID provides short-distance interaction information in the native complex inside the parasite, a topical model of BC architecture is within reach. To that end half the known BC components will be tagged as baits in BioID. Proof of principle experiments already generated several new insights underscoring the feasibility of this approach. Quantitative mass spectrometry data will be used to assemble a protein-protein interaction (PPI) map, which is expected to identify both clusters within the basal complex and nodes that make connections with many components. Clusters are expected to align with different compartments observed by (ultra)structural studies. Nodes will highlight potential key organizers of (sub)structures, which makes them good targets for functional studies. To maximize the depth of biological insights that can be realistically achieved under this proposal, 10 key candidates will be prioritized based on PPI map position and biological signature. Their spatiotemporal dynamics throughout parasite development and localization within the BC will be tracked by auto-fluorescent and/or epitope tags. Dynamical changes likely align with different assembly steps and/or functions of the BC. Furthermore, 5 candidates among the 10 primary picks representing as much diversity as possible will be selected for the generation of (conditional) gene knock-out (KO) strains. The KO strains will be evaluated for defects in BC assembly, morphology and constriction as well as IVN formation, morphology and function in uptake of host cell nutrients. Altogether, these data will help to resolve structure-function relationships and provide a hint at molecular interplay and mechanisms underlying the various BC functions. These insights will guide future mechanistic studies and development of specific new drugs.